Monocytes/macrophages play a central role in immune response by secreting inflammatory cytokines upon activation. Two of the major cytokines secreted by the activated monocytes are Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Cytokines stimulate immune cells by autocrine and paracrine mechanisms. Treatment with morphine seems to affect the functioning of the immune system by selective inhibiting the secretion of cytokines. At the present time the mechanism by which morphine inhibit cytokine gene expression is not completely understood. Based on our preliminary studies, we hypothesize that morphine inhibit the activation of an important transcription factor necessary for cytokine gene expression. In order to determine the molecular mechanisms involved, we will use a model system (Bac 1.25 cell line) representing monocyte activation by bacterial lipopolysaccharides (LPS). Morphine treatment of Bac 1.25 cells has been shown to inhibit LPS induced expression IL-6 and TNF-alpha. Expression of both of these cytokine genes is dependent on the activation of a transcription factor, NFkappaB. Interestingly, morphine treatment also inhibited the activation of NFkappaB by LPS. In the current proposal, we will investigate the molecular mechanism by which morphine selectively inhibit NFkappaB activation by LPS. Initially, we will study whether morphine inhibit NFkappaB synthesis or its translocation into the nucleus. NFkappaB is normally retained in the cytoplasm as an inactive complex with an inhibitory protein, IkappaB. Phosphorylation of specific serine and threonine residues induces a conformational change in IkappaB and dissociation in the complex. Phosphorylated IkappaB is a signal for its eventual ubiquitinylation and proteolytic degradation. We will systematically investigate how morphine treatment influences of IkappaB phosphorylation associated events. Finally, we will determine which of the upstream intracellular signaling pathways leading to NFkappaB activation is affected by morphine. These studies will help in understanding he molecular mechanism of morphine mediated immunosuppression and help in the development of novel therapeutic regimens to overcome opportunistic infections.